Activated leukocyte composition and uses for wound healing

ABSTRACT

Disclosed are therapeutic, blood-derived activated leukocyte compositions, methods of making them, and methods of using the compositions to repair or promote the prevention and healing of wounds.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of International ApplicationNo. PCT/IB2010/000882, filed Mar. 5, 2010, in English and designatingthe United States, which claims benefit of Provisional Application Ser.No. 61/209,298, filed Mar. 5, 2009, and Provisional Application Ser. No.61/211,587, filed Apr. 1, 2009, the disclosures of each of which arehereby incorporated herein by reference. This application also claimsbenefit of Provisional Application Ser. No. 61/381,268, filed Sep. 9,2010, and Provisional Application Ser. No. 61/460,024, filed Dec. 23,2010, the disclosures of each of which are also incorporated herein byreference.

BACKGROUND OF THE INVENTION

The wound healing process involves participation of white blood cells,also known as leukocytes. Leukocytes include lymphocytes, granulocytesand monocytes. Three common types of lymphocytes are T-cells, B-cellsand natural killer cells. T-cells and B-cells play important roles inthe recognition of antigens in the body (Parkin, 2001). Natural killer(NK) cells identify infected cells by alterations in the levels of themajor histocompatability complex (MHC), and destroy the infected cells(Moretta, 2008). The participation of lymphocytes in the healing processis largely associated with their production of cytokines and growthfactors (Keen, 2008). A new class of gamma-delta-T cells has beendescribed in the skin (Jameson, 2002. Havran, 2005). Among the differenttypes of granulocytes are neutrophils, basophils and eosinophils.Monocytes differentiate into macrophages, which are responsible fordestruction of tissue debris or invading foreign substances. Macrophagesalso produce molecules that control inflammation and repair (Riches,1996).

The process of wound healing occurs in three overlapping phases. (Li,2007; Broughton, 2006; Tsirogianni, 2006; Singer, 1999; Martin, 1997).The first phase is the inflammatory phase. It is characterized byrecruitment of neutrophils, followed by monocytes to the wound site,where they kill and phagocytize bacteria (Agaiby, 1999).

The second wound healing phase which is known as the proliferativephase, involves formation of new granulation tissue. Fibroblastsproliferate and migrate into the wound space and synthesize collagen andother components of extracellular matrix (Greiling, 1997). At the sametime, angiogenesis occurs, providing nutrients and oxygen to themetabolically active new granulation tissue (Tonnesen, 2000).Keratinocytes from the intact epidermis start to migrate over theprovisional matrix and begin to proliferate, leading the way for newepithelial tissue (Kim, 1992).

Remodeling is the third and final phase in wound healing. It ischaracterized by fibroblast differentiation into myofibroblasts, whichcontract and bring the wound edges closer together (Tomasek, 2002).Remodeling of the collagen fibers by degradation and re-synthesis allowsthe wound to gain strength by re-orientation of the collagen fibers (aprocess tightly controlled by growth factors) (Werner, 2003).

The challenge of treating wounds is often compounded by patients withmultiple pathologies such as diabetes, coronary artery disease andhypertension. These diseases have the common effect of exacerbatingvascular complications due to various physiological conditions.Complications from wounds may result in increased morbidity andmortality (Doshi, 2008).

Conventional wound treatments include surgical debridement, antibiotictherapies and various dressings (Moran, 2008; Fonder, 2008). Woundsresistant to conventional treatment are also referred to as refractorywounds. These wounds lead to a decrease in quality of life and canresult in increased morbidity and mortality. Thus, a need continues toexist for effective wound healing compositions and methods.

BRIEF SUMMARY OF THE INVENTION

One aspect of the present invention is directed to a method for makingan activated leukocyte composition (ALC) derived from blood (e.g.,obtainable or obtained from a whole blood sample). The method includesthe steps of subjecting leukocytes, which may be obtained from a sampleof whole human blood, to a first incubation for a period of time and ata temperature which allows the leukocytes to become activated, which inpreferred embodiments, is about 8 to about 20 hours, and at roomtemperature. After incubation, the leukocytes are contacted with aphysiologically acceptable aqueous solution such as sterile, distilledwater, to initiate hypo-osmotic shock, followed by contacting theshocked leukocytes with a physiologically acceptable salt solution torestore isotonicity. This activated leukocyte composition (ALC) may beused therapeutically. However, in some embodiments, separate andsubstantially concurrent with the first incubation of the leukocytes, asample of plasma, which may be obtained from the same or different wholeblood sample (i.e., from the same or a different human), is contactedwith a coagulating agent at about 37° C. concurrent with the leukocyteincubation, which in preferred embodiments, is about 8 to about 20hours, followed by separating serum from the coagulated plasma sample.The leukocytes are re-suspended in serum collected from the coagulatedplasma sample, thus forming the ALC. After the first incubation, theleukocytes may be further subjected to a second incubation for about 60to about 120 minutes at about 37° C.

Thus, in one aspect the invention provides a method for making anactivated leukocyte composition comprising: a) incubating humanleukocytes for a period of time so that the leukocytes transition from aquiescent to a functionally active state; b) subjecting the leukocytesto hypo-osmotic shock; and c) adding to the leukocytes of step b) a saltsolution in an amount which restores isotonicity. In some embodiments,the method further comprises mixing the activated leukocytes of step c)with serum.

In another aspect the invention provides a method for activatingleukocytes comprising: a) obtaining leukocytes from human blood; and b)incubating the human leukocytes for a period of time so that theleukocytes transition from a quiescent to a functionally active state.

In some embodiments of these aspects, the incubation of step a) occursat a temperature of about 12° C. to about 28° C. for a time ranging fromabout 8 to about 20 hours. In some embodiments, the incubation of stepa) occurs at a temperature of about 18° C. to about 24° C. for a timeranging from about 8 to about 12 hours. In other embodiments, theincubation of step a) occurs at a temperature of 12° C. to about 28° C.for a time ranging from about 90 minutes upwards of 2, 3, 4, 5, 6, 7, 8,or 12 to about 20 hours. In other embodiments, the incubation of step a)occurs at a temperature up to about 37° C. and for a time ranging fromfive hours to about 24 hours.

In some embodiments, the activated leukocyte composition has a shelflife extending up to 97 hours following collection of the humanleukocytes.

In some embodiments, the human leukocytes are obtained from a donorhaving an O negative blood type.

In some embodiments, the hypo-osmotic shock comprises contacting theleukocytes with water.

In some of those embodiments further comprising mixing the activatedleukocytes with serum, the serum is prepared from plasma obtained from adonor having AB positive blood type. Also, in some of those embodimentsfurther comprising mixing the activated leukocytes with serum, themixture of activated leukocytes and serum is incubated at about 37° C.for about 8-20 hours. In other embodiments, the mixture of activatedleukocytes and serum is incubated for about 60 to about 120 minutes.

In other aspects, the invention provides a composition comprisingactivated leukocytes made according to any of the methods of theinvention described herein.

Another aspect of the present invention is directed to an ALC derivedfrom blood. The activated leukocyte composition of the present inventionincludes, in terms of the population of leukocytes present therein,about 40% to about 90% granulocytes, about 5% to about 20% monocytes andabout 5% to about 30% lymphocytes, based on the total number ofleukocytes in the ALC. As shown in the working examples, the inventiveALCs may also be characterized and distinguished from known compositionsin terms of minimum yield of leukocytes (relative to the whole bloodsample), viability of leukocytes, and minimum activation levels ofgranulocytes, e.g., as indicated by CD11b. The ALC may further containresidual levels of platelets (in amounts of about 46.8+/−39.2(10³/μl)and red blood cells (in the amount of about 0.1+/−0.06(10⁶/μl) of theALC. The population of granulocytes may include about 52% to about 78%neutrophils; about 1% to about 9% eosinophils; and about 1% to about 2%basophils. The population of lymphocytes may include about 7% to about25% B cells (CD19+), about 20% to about 30% NK cells (CD3−/CD56+), about40% to about 60% T cells (CD3+), about 0%, for example, about 0.1%, toabout 30% of NKT cells CD3+/CD56+, about 8% to about 20% of T helpercells (CD4+/CD3+), and about 20% to about 30% of CD8+/CD3+ cells.

In some embodiments, the composition includes T-helper cells andT-suppressor cells in a ratio of less than 0.8.

In some embodiments, the composition further comprises mesenchymal stemcells in an amount ranging from about 0.1% to about 5.0% of the totalcell population in the composition.

In some embodiments, the composition further comprises endothelialprogenitor cells in a amount ranging from about 0.1% to about 5.0% ofthe total cell population in the composition.

The cells may be suspended in a carrier such as serum (which may beautologous or allogeneic with respect to recipient) or some otherphysiologically acceptable iso-normal liquid suitable for storing andadministering cells, such as the solution used to restore isotonicity.

Another aspect of the invention relates to articles of manufacturecomprising a composition of the invention and a dressing. In someembodiments, the dressing is a dry dressing, moisture-keeping barrierdressing, or bioactive dressing. In those embodiments involving a drydressing, the dressing may be a gauze, a bandage, a non-adhesive mesh, amembrane, foils, foam, or a tissue adhesive. In those embodimentsinvolving a moisture-keeping barrier dressing, the dressing may be apaste, a cream, an ointment, a nonpermeable or semi-permeable membraneor foil, a hydrocolloid, a hydrogel, or combinations thereof. In thoseembodiments involving a bioactive dressing, the dressing may be anantimicrobial dressing.

In still another aspect, a composition of the invention may furthercomprise as a matrix or scaffold a material suitable for implantation ina person. In some embodiments, the material is a solid beforeimplantation. In some embodiments, the material is a gel that solidifiesfollowing implantation.

Yet another aspect of the present invention is directed to a method ofpromoting wound healing or of treating a wound, which includesadministering or otherwise applying the ALC to a wound. In someembodiments, the wound is a decubital ulcer, a pressure ulcer, a lowerextremity ulcer, a deep sternal wound, a post-operative wound, arefractory post-operative wound of the trunk area, a wound to the greatsaphenous vein following harvesting of the great saphenous vein, avenous ulcer, or an anal fissure. In those embodiments involving a lowerextremity ulcer, the ulcer may be in a diabetic patient. In otherembodiments, the wound is a venous ulcer, pressure ulcer, orpost-operative ulcer.

In other aspects, the invention is directed to a method of inhibitingthe onset of infection in a wound, comprising administering to the wounda composition of the invention. In one embodiment, the wound is causedby trauma. In another embodiment, the wound is caused by surgery.

In those aspects involving methods of treating a wound, the wound mayalso be treated by administering to the wound an article of manufacturecomprising a composition of the invention.

For purposes of the present invention, wounds include tattoos.Accordingly, a further aspect of the present invention is directed to amethod of tattoo removal, which includes administering or otherwiseapplying the ALC or an article of manufacture comprising the ALC, to atattoo.

The disclosed invention achieves several unexpected results compared toat least one known wound healing composition containing white bloodcells. As demonstrated in working examples herein, these results includeincreased yield and viability of leukocytes (WBCs), higher percentage ofactivated granulocytes, and the presence of unexpectedly highconcentrations of mesenchymal stem cells and endothelial progenitorcells. The disclosed invention is also believed to include anunexpectedly and relatively high percentage of activated monocytes(compared to blood) and a relatively higher percentage of CD8 T-cellscompared to CD4 T-cells.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 schematically depicts a first portion of a representative systemfor producing the ALC compositions of the present invention, whichincludes bags A-C (Set-1), which are blood storage bags or containers,wherein bag A contains packed red blood cells collected from a donor;bag B contains plasma; and bag C contains leukocytes (which followinginitial separation from whole blood form a layer commonly referred to asthe buffy coat).

FIG. 2A schematically depicts a second portion of the representativesystem for producing ALC compositions of the present invention, whichincludes a 7-bag set after bag A with RBC is removed from the system andbags B and C from FIG. 1 are welded to bags 1-5 (Set-2); and FIG. 2Bshows a 6-bag set that may be used in lieu of bags 1-5 in FIG. 2A.

FIGS. 3A and 3B are graphs illustrating the general trends of both CD62Land CD42b expression, which are indicators of cell activation.

FIGS. 4A-4E demonstrate the results of identification of mesenchymalstem cells (MSC) in ALS using flow cytometry analysis. FIGS. 4A-4D aredot plot graphs illustrating the sequential steps of gradually narrowing(gating) cell population containing MSC, ending with a practicallyhomogenous (in terms of size and surface character) population of cellsnegative for the common marker of leukocytes (CD45) and bearing MSCmarker CD105. FIG. 4E depicts statistical analysis of sequentially gatedpopulations, demonstrating that the final population of CD 105positive/CD45 negative cells composes 2.7% of CD105 highly positivecells and 0.2% of all cells in the tested ALS sample.

FIGS. 5A and 5B demonstrate the results of identification of endothelialprogenitor cells in ALS using flow cytometry analysis. A dot plot graphdepicted on FIG. 5A, shows distribution of ALS cells stained withantibodies against two markers of endothelial progenitors, CD31 and KDR,according corresponding fluorescences. The upper right quadrant of thedot plot contains cells double-positive for both markers. Statisticalanalysis showed that this CD31^(positive) _(/KDR) ^(positive) cellpopulation composes 0.2% of all cells in ALS. FIG. 5B depicts a controlexperiment: a similar dot plot graph, where cells of ALS were stainedwith irrelevant but isotype-matched antibodies. No positively-stainedcells were found.

DETAILED DESCRIPTION OF THE INVENTION

Blood is defined herein as whole blood or any of its constituent parts(e.g., plasma, leukocytes, platelets or red blood cells). The amounts ofplatelets and red blood cells that may be present in the ALC of thepresent invention may be lower than that in whole blood.

The term “about” as used herein in connection with any and all values(including lower and upper ends of numerical ranges) as any value havingan acceptable range of deviation of +/−0.5% to +/−20% (and valuestherebetween, e.g., ±1%, ±1.5%, +2%, ±2.5%, ±3%, ±3.5%, ±4%, ±4.5%, ±5%,±5.5%, +6%, ±6.5%, ±7%, ±7.5%, ±8%, ±8.5%, ±9%, ±9.5%, ±10%, ±10.5%,±11%, ±11.5%, ±12%, ±12.5%, ±13, ±13.5%, ±14%, ±14.5%, ±15%, ±15.5%,+16%, ±16.5%, +17%, +17.5%, ±18%, ±18.5%, ±19%, ±19.5%, and ±20%).

The starting materials for producing the inventive ALCs may be obtainedfrom several sources. Whole blood or one or more components thereof(e.g., leukocytes and plasma) may be obtained from autologous orallogeneic sources. In one embodiment of the present invention, theblood sample is collected from the patient who will ultimately betreated with the ALC, which is referred to herein as an autologous bloodsample or source. In embodiments wherein the source(s) i.e., the bloodor its components, is obtained from an individual other than theintended ALC recipient, which is referred to as an allogeneic bloodsample or source, these starting materials may be conveniently obtainedfrom a blood bank. The samples may be screened by the blood bank forblood type (ABO, Rh), irregular antibodies to red cell antigens, andtransfusion-transmittable diseases. More specifically, screening can beconducted with antibodies using an Abbott PRISM instrument against:Hepatitis B, C, HIV ½, HTLV and Syphilis (−HCV; HbsAg; anti-HIV ½ O+;and anti-HTLV I/II). The samples can also be screened for HIV, HCV andHBV by molecular methods (NAT-Nucleic Acid Testing). Molecular screeningcan be accomplished using commercially available instrumentation, e.g.,the TIGRIS system of Chiron.

In these embodiments involving allogeneic sources, the samples can beobtained from donors with the same blood type as the intended ALCrecipient. Alternatively and as further described herein, plasma samplescan be obtained from donors with AB+ blood and the leukocytes can beobtained from patients with O− blood. Patients with AB+ blood areuniversal donors for plasma and patients with O− blood are universaldonors for leukocytes. In still other embodiments, the leukocytes and/orplasma may be of any blood type. The plasma used in the invention can befresh, stored (e.g., at 1-6° C. for less than 24 hours), dried, orotherwise pre-treated (e.g., pathogen-reduced plasma andsolvent/detergent (SD) treated plasma). The plasma can be fresh orstored at 1-6° C. for less than 24 hours, or Fresh Frozen Plasma, orDried Plasma, or Pathogen-Reduced Plasma, or Solvent/Detergent (SD)Treated Plasma. Regardless of the source, all necessary processing ofthe sample(s) can be carried out without the need for highly specializedequipment.

A preferred method of making the ALC composition of the presentinvention is now described with reference to FIGS. 1 and 2, whichillustrate a system containing two sets of interconnected sterileinfusion bags. The system is sealed so that there is no exposure to theoutside environment. Specifically, the tubes connecting the two sets arewelded together to form one system using a Sterile Connecting Device(e.g., TSCD®-II Cat number ME-203AH of Terumo). More specifically, toensure compliance with sterility standards, the welding and cutting ofthe tubes is done by pre-heating special wafers, typically at about 300°C. (although sterility can be effectively achieved by pre-heating atlower or even higher temperatures). This high temperature increases thesterility of the welding procedure. To further ensure sterility, thewelding may be performed in a class 100 Biological Safety Cabinet withina class 100,000 containment area.

As illustrated in these figures, the system contains two sterile bagsets. Set 1, containing bags A, B, and C, is a standard, commerciallyavailable triple bag set commonly used for blood transfusion. A humanblood sample, typically in the volume of about 400 to about 550 ml, iscollected in a blood bank via venipuncture and placed into bag A, andthen fractionated into its component parts using standard techniquesinto bags A, B and C. For example, bag A containing the blood sample iscentrifuged. After centrifugation, the blood components are separated,e.g., using a blood component extractor manufactured by Baxter. Thebuffy coat containing leukocytes is placed into bag C, plasma is placedinto bag B and erythrocytes remain in bag A. Thus, as a result of thisprocess, bag A contains packed erythrocytes; bag B contains plasma; andbag C contains the buffy coat containing leukocytes (and possiblyresidual plasma and erythrocytes). Alternatively, the blood componentscan be separated from whole blood via apheresis techniques known in theart.

Bag A is then disconnected from the three-bag set. As illustrated inFIG. 2, bags B and C are then welded to custom made infusion bags 1-5(Set-2) to form the system used to make the activated leukocytecomposition. As described in these embodiments, bags 1-5 have volumes of500 ml, 50 ml, 50 ml, 100 ml and 500 ml, respectively. As disclosedabove, welding is performed with a sterile connecting device.

Bag 1, which is used for both first and second incubations of theleukocytes, contains 200 ml of sterile filtered air. If bag 1 isgas-permeable, there will be no need for air bags. Gas-permeable bagsmay also be treated or otherwise modified so that they become adhesivefor leukocytes. For example, the bags may contain scaffolds or leukocyteagonists such as complement protein, interferon-alpha, interferon-gammaand interleukin-12. Leukocyte adhesion to the bag surface could bebeneficial for their ability to release soluble agonists. The bags couldbe made from adhesive plastic or regular plastic treated in such a wayas to become adhesive (corona discharge, liquid gas plasma, etc.), orcoated with extracellular matrix proteins or chemically modified. Thescaffolds may be in different shapes and in particular could bemicrobeads, biodegradable or not biodegradable, e.g., made of collagen(or fragments of collagen, wherein the collagen or collagen fragmentsare mammalian in origin, such as human or bovine-derived collagen orfragments thereof), or made of PLA, PGA (polylactic acid, polyglycolicacid) or similar synthetic polymers, hydrogel scaffolds made of gelatin,hyaluronic acid alginate or fibrin sealer. Scaffolds could be coatedwith adhesion receptors, extracellular matrix proteins such asfibronectin or laminin or with active binding peptides fromextracellular matrices, such as RGD. Scaffolds or microbeads could bealso coated with activating stimuli or stimulating antibodies (thestimuli that otherwise are not desirable in the product will beeliminated together with scaffolds at the end of the productionprocess).

Bag 2 contains a solution (e.g., 20 ml of buffered sodium chloridesolution (8.91% NaCl, USP), or any other physiologically acceptablesolution containing inorganic ions, organic osmolytes such as sucrose,or some combination thereof, such as Lactated Ringers (Hartmans)solution), which serves to restore the leukocytes to isotonicityfollowing hypo-osmotic shock. When the sodium chloride solution is addedto 200 ml of distilled water (in bag 5), it becomes a 0.9% NaClsolution. Bag 3 contains 20 ml of sterile filtered air. Bag 4 contains asolution (e.g., about 60 ml of buffered calcium chloride solution (1.17%CaCl₂ dihydrate, USP), which acts to coagulate the plasma in bag B, andto facilitate separation into platelets and serum. Bag 5 contains about200 ml water.

The set is packed as a single unit and sterilized using high pressuresteam, which greatly reduces the risk of secondary infection to thepatient.

The leukocytes are then transferred from bag C into bag 1 and incubatedwhile the bag is maintained in a vertical or in a flat position andunder activating conditions including time and temperature, to allowthem to become activated. For purposes of the present invention,leukocyte activation is defined as a process involving at least onestage, by which the cells (leukocytes) undergo a transition from aquiescent to a functionally active state which is accompanied bysynthesis of biologically active substances or translocation ofpre-synthesized substances, e.g., cytokines including IL-8, from thecytoplasm to the cellular membrane or their release into extracellularmedium (which in this case is serum). Activation of leukocytes in vivomay involve migration of the cells closer to and along the blood vesselwall, which is mediated by P-selectin (and increased CD42b expression),increased adhesion of leukocytes to the endothelial wall, spreading andextravasation, which is mediated to a large degree by activated CD11bthat interacts with endothelial ligands ICAM-1 and ICAM-2; migration tothe focus of inflammation via interaction with extracellular matrixproteins e.g., laminin) and functional responses to inflammatory stimulisuch as respiratory burst, degranulation, phagocytosis and release ofcytokines. For purposes of the present invention, activation of theleukocytes, at least as a result of the first incubation, may beindicated by increased expression of activated form of CD11b receptor onleukocyte populations including granulocytes, monocytes and lymphocytes,and higher expression levels of CD69, a lymphocyte-specific activationmarker. In some embodiments, leukocytes that have undergone the firstincubation may also exhibit increased levels of CD69, alymphocyte-specific activation marker, express platelet marker 42B (as aresult of the interaction between activated granulocytes and monocyteswith residual platelets in the buffy coat via p-selectin) and/orincreased production of IL-8. Yet other indicia of leukocyte activationmay include increased production of one or more of proteins orpolypeptides, lipids, sugars, oxygen radicals and other biochemicalmoieties that function as adhesion molecules, cytokines in addition toIL-8, growth factors, enzymes, transcription factors and cell signalingreceptors and mediators. Altered expression levels of any of thesemolecules is assessed from the standpoint of the leukocytes contained ina “fresh buffy coat” (as described herein), without being subjected toan incubation. Once the leukocytes are activated, they remain activatedand as described herein, as a result of subsequent steps in theinventive methods, may achieve higher levels of activation, e.g., evengreater expression levels of CD11b; greater or even greater expressionlevels of CD69, and even lower expression levels of CD62L. Thus, thefirst and (the optional) second incubation, and the intervening steps,may be collectively referred to as the “activation process”.

In some embodiments, the leukocytes are incubated simply by allowingthem to stand at room temperature. For purposes of the presentinvention, room temperature refers to a temperature in the range ofabout 12° C. to about 28° C., and in some embodiments from about 16° C.to about 25° C., from about 18-25° C. and from about 20-25° C. The timeperiod of incubation, which may vary depending upon the temperature,generally ranges from about 30 minutes to about 24 hours. The incubationtime needed to activate the leukocytes will be roughly inverselyproportional to the temperature at which the incubation is conducted.Thus, incubation times will be lower at increased temperatures. Forexample, in embodiments where leukocytes are allowed to stand at roomtemperature, the incubation time generally ranges from about 90 minutes,and upwards of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 21, 22, 23 or upwards of 24 hours (and subranges thereofwhich include, for example a minimum time of anywhere from 90 minutes, 2hrs, 3 hrs, 4 hrs, 5 hrs, 6 hrs, 7 hrs, 8 hrs, 9 hrs, 10 hrs, 11 hrs, 12hrs, 13 hrs, 14 hrs, 15 hrs, 16 hrs or higher). In preferredembodiments, the incubation time at room temperature ranges from about3, 4, 5, 6, 7 or 8 hours to about 20 hours. In a more preferredembodiment, incubation of the leukocytes occurs at about 18° C. to about24° C. for about 8 hours to about 12 hours. In other embodiments,incubation of the leukocytes involves exposing them to heat, e.g., at atemperature above room temperature and up to about 37° C. The timeperiod for incubation at elevated temperatures generally ranges anywherefrom 30, 45, 60, or 90 min to about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12hours (and even upwards, in hour increments to about 24 hours (andsubranges thereof which include, for example, a minimum time of anywherefrom 30, 45, 60 or 90 mins, 2 hrs, 3 hrs, 4 hrs, 5 hrs, 6, hrs, etc.).

After incubation, the leukocyte suspension (including any cells thatadhere to the bag during the first incubation which will immediatelydetach after addition of water) is subjected to hypo-osmotic shock. Inpreferred embodiments, hypo-osmotic shock is performed immediately(i.e., upon completion of the preceding step without any interveningstep or unnecessary delay, typically less than 2 minutes). Thehypo-osmotic shock may be initiated by transferring the distilled waterfrom bag 5 to bag 1 containing the leukocytes. The hypo-osmotic shocktreatment is typically conducted for about 25-45 seconds. Lesser timeswithin this range are preferred, as it is believed that fewer CD4+ Tcells are lost. CD4+ T cells are known to produce various cytokines(e.g., IFN gamma, IL-2, IL-4 IL-17), which might be beneficial for woundhealing. Following this step, and preferably immediately thereafter,isotonicity is restored to the leukocytes by transferring the sodiumchloride solution from bag 2 to bag 1. The ratio of the volume of sodiumchloride solution to cell suspension in water is generally about 1:10.

Following the treatment that restores isotonicity, the entire 7-bagsystem (now containing only 5 bags) is centrifuged. This process removeswater and salt solution added in the course of the two prior steps, andprevents exposure of the leukocytes to hemolysate (of the erythrocytes).As a result of the centrifugation, the leukocytes form a pellet. Aftercentrifugation, the supernatant from bag 1 is transferred into bag C,and the leukocyte pellet formed in bag 1 as a result of thecentrifugation is re-suspended in serum. As described above, thiscomposition may be used therapeutically in the inventive methods.

The preferred embodiment involves at least one additional step. Thus, ina separate step, which may be conducted concurrently with the leukocyteincubation, plasma is separated into platelets and serum by the use of acoagulant such as CaCl₂. This process results in formation of a bloodclot of predominately fibrin strands and platelet aggregates. The bloodclot is separated from serum, which is essential plasma depleted ofclotting proteins and most of the platelets. As a result of thistreatment, the resulting serum has a residual platelet level thatgenerally ranges from about 0 to about 0.2×10³ per μL Thus, in thisrepresentative embodiment, CaCl₂ from bag 4 is transferred to bag B. BagB, which now contains a composition of plasma and CaCl₂, is typicallyallowed to coagulate at a temperature of about 37° C. The plasma remainsin contact with the coagulating agent for substantially the same periodof time the leukocytes are incubated. In this preferred embodiment ofthe present invention, the activated leukocytes are then incubated inthe coagulated plasma before the final composition is made. Generally,this second incubation period is conducted for about 1-2 hours at 37° C.

It should be noted that although the plasma and the serum for use invarious aspects of the invention may be prepared at the same time as theactivated leukocyte composition (whether from the same or a differentblood sample), in alternate embodiments the serum and/or plasma isprepared independently of the activated leukocyte composition. Forexample, serum and/or plasma may be obtained from a commercial ornon-profit supplier of blood products. The serum and/or plasma may berecently obtained from one or more donors, or it may have been storedfor a period of time, and may be stored frozen. Further, since the bloodsamples used to prepare the serum and/or plasma need not be the same asused to prepare the activated leukocytes (whether or not they are fromthe same donor), their preparation need not occur at the same physicallocation or at the same time as preparation of the activate leukocytecomposition.

In an alternative and even more preferred embodiment, the 6-bag setillustrated in FIG. 2B may be used in place of bags 1-5 illustrated inFIG. 2A. The additional bag helps maintain sterility of the clean roomproduction site. In this embodiment of the present method, afterseparation into buffy coat, plasma and RBC, leukocytes as previouslydescribed are transferred from bag C to a sterile bag 1 and the plasmais placed into the sixth bag which is also sterile, and then the 6 bagsystem is separated from the original triple bag set.

In other embodiments, the ALC composition may be prepared from smallervolumes of blood samples, with commensurate decreases in volumes of allsolutions and use of smaller bags. Furthermore, use of these differentsize bags yield ALCs with different compositions. Even in theseembodiments, allogeneic or autologous blood samples may be used asstarting materials. Use of smaller volumes provides the clinician withthe ability to perform the blood collection autonomously, without usingan external blood bank, such as in emergency situations in treatingpatients with otherwise healthy immune systems but suffering from sometype of traumatic wound (e.g., battlefield and combat conditions). Inthese embodiments, testing for transmittable diseases and antigens maybe dispensed with. However in such cases, patients with refractorywounds are not clinically acceptable blood donors for effective ALCpreparation. When this situation arises, ALC will be produced fromallogeneic donors by the means described herein.

The ALCs of the present invention include leukocytes, e.g.,granulocytes, monocytes and lymphocytes. Granulocytes includeneutrophils, eosinophils and basophils. In its broadest sense, theleukocyte population of the ALC generally contains about 40% to about90% granulocytes, about 5% to about 20% monocytes and about 5% to about30% lymphocytes. Specific amounts of the cells may differ based on theanalysis techniques employed. When analysis is performed using FACS(e.g., using a side-scatter versus a forward-scatter dot plot analysis),the leukocyte composition generally contains about 55% to about 80%granulocytes; about 5% to about 15% monocytes and about 5% to about 30%lymphocytes, and in some embodiments, comprises about 58-76%granulocytes; about 5-11% monocytes and about 9-23% lymphocytes. Whenanalysis is performed using a Cell Dyn Analyzer, the leukocytecomposition generally contains about 50% to about 90% granulocytes;about 5% to about 15% monocytes; and about 10% to about 25% lymphocytes.The subpopulation of lymphocytes in the ALC may confirm the followingcells in the general ranges as follows: about 7% to about 25% B cells(CD19+); about 20% to about 30% NK cells (CD3−/CD56+), about 40% toabout 60% T cells (CD3+); about 0.1% to about 30% of NKT cellsCD3+/CD56+, about 8% to about 20% of T helper cells (CD4+/CD3+), andabout 20% to about 30% of CD8+/CD3+ cells. In preferred embodiments, thelymphocyte subpopulation is enriched with at least 9% CD56+ cells(CD3−/CD56+; CD3+/CD56+; CD3+/CD56+/CD8+), the amount of the T helperlymphocytes (CD4+/CD3+) is decreased to less than 20%, and/or the ratioof T-helper to T-suppressor cells (CD4+/CD3+: CD8+/CD3+) is less than0.8. The ALC of the present invention may also include stem andprogenitor cell populations. For example, the ALC may further containmesenchymal stem cells and endothelial progenitor cells, each in amountsranging from about 0.1% to about 5.0% of the total cell population inthe ALC.

In one embodiment, the ALC has a shelf life (i.e., can be stored priorto use) ranging up to 72 hours following production, for example about5, 10, 15, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more hoursfollowing its production.

In one embodiment, the ALC has a shelf life extends up to 97 hoursfollowing collection of the blood sample or blood samples used toprepare the ALC, that is about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55or more hours following collection of the blood sample or blood samplesused to prepare the ALC.

Patients suffering from wounds can be physiologically compromised orotherwise healthy. For example, due to already impaired metabolicsystems, diabetics and other medically compromised patients arecandidates for ALC derived from heterologous blood, as their ownleukocytes may not be optimal for the procedure. However, otherwisehealthy patients, as in the example of trauma patients, are also goodcandidates for ALC compositions of the present invention.

The present invention is useful in promoting healing in a multitude ofwound types. Although in practice it may be used in combination withother treatment modalities, it does not require them to achieveeffective wound healing. The inventors have contemplated application ofthe ALC to any type of wound and foresee no limitations as to the typeof wound that can be treated. The ease of application, e.g., with astandard syringe or similar application device, makes the inventive ALCcompositions safe and easy to use.

Wounds amenable to treatment with the invention are typically in thefaun of burns, punctures, and cuts or tears of the living tissues.Wounds of the skin can penetrate the epidermis, dermis or in the case offull-thickness wounds, the subcutaneous tissue. Thus, representativetypes of wounds amenable to treatment with the compositions and methodsof the present invention include burns (e.g., caused by exposure to fireor an agent that is highly caustic to skin such as radiation), ulcers(e.g., decubital or pressure ulcers; venous ulcers and diabetic ulcers),deep sternal wounds, e.g., following open heart surgery (to the greatsaphenous vein after coronary revascularization and harvesting of thegreat saphenous vein); and post-operative wounds following abdominal,orthopedic and any other types of surgery. Other wounds are those whichresult from trauma such as incurred during combat or other violentactivity, including wounds caused by gun shots, knives, or any otherobject able to cause a cut or tear in the skin. Wounds of the oralcavity (e.g., teeth), as well as wounds that arise as a side-effect ofmedication or as a symptom of various pathologies (e.g., soresassociated with Kaposi's Sarcoma), as well as internal wounds (e.g.,ruptures of muscle tissue such as anal fissures), and wounds or lesionsto the gastrointestinal tract, such as ulcers in the stomach orintestines) may also be amenable to treatment with the presentinvention. In addition to providing a wound-healing effect, the ALC ofthe present invention may also provide a benefit of inhibiting onset ofinfection following such therapy, particularly in the context ofsurgery.

The ALC of the present invention may also be useful to remove tattoos.Without intending to be bound by theory, it is believed that theactivated granulocytes and neutrophils engulf and degrade the ink.

The ALC may also be used to treat any wounds exacerbated by vascularinsufficiency. Vascular insufficiency, for purposes of the presentinvention, refers to inadequate blood circulation resulting ininsufficient perfusion to the afflicted areas. Such insufficiency can becaused by trauma (e.g., damage to the vasculature adjacent to a skeletalfracture), or various pathologies (e.g., diabetes and atherosclerosis).In either instance, whether trauma or disease induced, vascularinsufficiency decreases the likelihood of effective wound healing. TheALC may be useful in improving wound healing outcomes in these patientsand should be administered according to the methods described herein.Additionally, treatment algorithms should not be limited by the severityor type of wound, or the extent of vascular insufficiency. ALC may bemore efficacious in patients presenting with the most severe wounds andvascular insufficiency.

In general, application of the activated leukocyte composition isaccomplished by means of one or more injections of the ALC at a singleor multiple sites, using a suitable syringe (e.g., a 2 ml syringe fittedwith an 18 G or 25 G needle) directly into the wound or the tissuesurrounding the wound. In some embodiments, injection occurs about everyone centimeter to about every three centimeters for the entire length ofthe wound. At each injection site, about 0.1 to about 0.3 ml of ALC isinjected. ALC compositions of the present invention contain leukocytesin a concentration that generally ranges from about 2×10⁶ cells/ml toabout 4×10⁶ cells/ml.

For injection into the wound, it is preferred to use a Luer-Lock syringeor any other commercially available syringe that has a locking mechanismbetween the syringe and the needle. The biological space of a wound,particularly a pressure wound, is often limited. When injecting into awound, there is a risk of pressure causing the syringe to separate fromthe needle. Using a locking syringe eliminates this risk.

When injection into the wound tissue is not possible, the ALC can beapplied directly into the cavity of the wound. Application in thismethod can be done using direct application with a syringe or tubing.

The ALC may be applied to or around the wound site with the aid of adressing. Dry dressings include gauze and bandages, non-adhesive meshes,membranes and foils, foams, and tissue adhesives. Moisture-keepingbarrier dressings include pastes, creams and ointments, nonpermeable orsemi-permeable membranes or foils, hydrocolloids, hydrogels, andcombination products. Bioactive dressings include antimicrobialdressings, interactive dressings, single-component biologic dressings,and combination products (e.g., ointments, gels, fibrin sealant, growthand angiogenic factors (e.g., PDGF, BEGF, collagen)). In someembodiments, the wound is packed with sterile gauze soaked in the ALC.The dressing, such as sterile gauze pads, may be saturated withcompositions such as Lactated Ringer (Hartman) Solution, alginatecontaining dressing, polyurethane dressing or carboxymethylcellulosedressing, which is applied to cover the wound, followed by applicationof dry dressing. If the subject wound is highly infected, then silverdressings such as Silverlon can be applied. The choice of post-injectiondressing is based on the determination of the clinician. Commercialavailability, history of past clinical success, and patient toleranceare all factors to be considered in the selection of a wound dressing.The dressing may be removed periodically, e.g., typically after about 24hours, in order to irrigate the wound e.g., with sterile water and soap.

In another embodiment, the ALC composition is combined with aphysiologically inert and/or resorbable matrix or scaffold prior toadministration. The matrix or scaffold may be formed from any materialsuitable for implantation into a person. For example, the matrix orscaffold may comprise any biocompatible material including collagen,hyaluronic acid, or gelatin, or combinations thereof. The collagen maybe obtained from any source, including collagen prepared from humantissue or the tissue of other collagen-producing mammals. Thecombination of the ALC composition and matrix/scaffold material may forma gel or putty that is administered to a person by means of a press fit,or by injection. This allows for a sustained delivery of the ALC intothe site which benefits the patient in that the cells have a longerperiod in situ. In some embodiments, the mixture of the ALC compositionand matrix/scaffold material is prepared commercially and provided to ahealth-care provider pre-mixed. In other embodiments, the health careprovider mixes the ALC composition and the matric/scaffold prior toadministration to a person.

The ALC compositions may be applied to the wound once or more than once,e.g., after 4 weeks, once a clinician determines whether anotherapplication is necessary. Factors that may be taken into account includeincreased wound dimensions (width, length and depth), suppuration,pyrexia or any other sign or symptom indicating a recalcitrant infectionsuch that re-treatment is warranted. In addition to re-treatment,referral for surgical debridement may be indicated at any point theclinician deems appropriate.

The ALC may be used in conjunction with any other conventional woundtreatment, such as negative pressure, warming (therapeutic heat),electrical stimulation, magnetism, laser phototherapy, cycloidalvibration therapy and ultrasound. It also can be used with biologicaltherapy such as larva therapy, skin substitutes, cultured keratinocytes(Epicel, Genzyme biosurgery), human dermal replacement (Dermagraft,Smith and Nephew Inc.), cadaver derived processed dermis (Allodeim, LifeCell Corporation), Bilayered Skin Equivalent (Apligraf, OrganogenesisInc.), TransCyte (Smith and Nephew Inc.), Growth Factors (PDGF iscurrently the only growth factor licensed for topical use), and fibrinsealant. In some embodiments, the ALC is used in conjunction withnegative pressure wound therapy (NPWT) (one example being the V.A.C.,which is a commercially available wound therapy manufactured by KCl).Negative pressure therapy promotes wound healing by applying negativepressure to a wound. In these embodiments, ALC is preferably applied toa wound prior to negative pressure therapy. In yet other embodiments,the ALC is used in conjunction with hyperbaric oxygen therapy (Thackham,2008) or ozone therapy. For example, the ALC can be applied to a woundjust prior to a patient receiving hyperbaric therapy. The ALC may alsobe used in conjunction with low-energy shock wave therapy (e.g.,impulses of about 0.1 mJ/mm²; 5 Hz) per centimeter of wound length).See, e.g., Dumfarth, et al., Ann. Thorac. Surg. 86:1909-13 (2008).

After treatment, the wounds may be evaluated for length, width andheight measurements. Typically, a wound is considered healed when allmeasurements of these parameters are negligible. The ALC may alsoprovide an analgesic effect.

The activated leukocyte composition is particularly useful in woundsincluding diabetic foot ulcers and decubital ulcers. Decubital ulcersare pressure ulcers caused by impeded blood flow, usually due toprolonged pressure on a particular area. (Berlowitz, 2007) Decubitalulcers cause morbidity and mortality in elderly people. At least 48% ofstage 1V pressure ulcers remain unhealed after one year of treatment.(Girouard, 2008). Patients suffering from decubiti also commonly haveco-morbid pathologies such as diabetes and hypertension. Thesepathologies further complicate the successful treatment of decubiti.

In one embodiment for treating decubital ulcers, the composition isaspirated into a sterile syringe of any size, using an 18-gauge (18G)needle. Aspiration is performed slowly to minimize damage to the cells.While the size of the syringe and needle are by no means limiting, alarge gauge needle is preferred for aspiration. This facilitates thetransfer and reduces cell damage.

Application of the activated leukocyte composition to the ulcercomprises injecting the composition into the wound. The entire sample inthe syringe can be deployed and the clinician can choose to administeradditional ALC if it is determined to be necessary based on clinicalparameters.

The 18G needle used for aspiration is exchanged with a needle ranging insize from 22-35 G. The ALC may be injected into the wound in variouslocations. In one embodiment, injection occurs about every onecentimeter to about every three centimeters for the entire length of thewound. At each injection site, 0.1-0.3 ml of ALC is injected.

In another embodiment, the entire syringe can be injected at one timeinto a single site within the wound.

Aspect(s) of the present invention will now be described in accordancewith the following non-limiting examples.

EXAMPLE 1 Analysis of Cellular Activation

An activated leukocyte composition made in accordance with the preferredembodiment of the present invention was quantified by the analysis ofvarious cell surface markers. An increase in platelet interaction witheither monocytes or granulocytes through the expression of P selectin isa sign of activation of the monocytes and granulocytes. Because residualplatelets remaining in the buffy coat adhere to activated granulocytesand monocytes the latter exhibit platelet marker CD42b on their surface.

CD62L is an adhesion receptor from a selectin family. It isconstitutively expressed on all classes of leukocytes includinggranulocytes, monocytes and lymphocytes. Upon activation, leukocytesrapidly shed off CD62L from their surface. CD62L is a plasma membraneprotein which is shed during activation and thus decreases with cellactivation. CD42b is a platelet activation marker involved in theprocess of coagulation as an aggregating factor. It interacts withextra-cellular matrix as well as with adhesion molecules and also usedin the present invention as an indicator of monocyte and granulocyteactivation.

Leukocytes were sampled at three time points: immediately prior to thebeginning of the production process (fresh buffy coat (FBC)); rightafter the first incubation (incubated buffy coat (IBC)); and from finalactivated leukocyte composition or final product (FP), meaning afterhypo-osmotic shock, followed by 90 min incubation with serum at 37° C.At each time point, leukocytes were labeled with specific monoclonalantibodies against corresponding activation markers and then analyzed byflow cytometry.

For antibody staining, cells from each time point were washed with FACSstaining solution (PBS, 2% Normal Mouse Serum; 0.02% Sodium Azide),aliquoted at 0.5×10⁶/tube and incubated with appropriate monoclonalantibodies for 30 min in the dark. After incubation, the cells weretreated with erythrocyte lysis buffer, washed, re-susupended in PBS andanalyzed on FACSCalibur flow cytometer (Becton Dickinson). In order tobetter distinguish between leukocyte populations, cells labeled withCD62L or CD42b were also double-labeled with the antibody against amonocyte marker CD14 conjugated to allophycocyanin (APC). Cells stainedwith irrelevant but isotype-matching antibodies (together with anti-CD14 antibodies) under the same conditions were used as negative controlsFor CD62L positive (+) staining cell suspension was incubated withanti-human CD14 (APC) and anti-human CD62L (FITC) antibodies at +4° C.For CD42b+ staining cell suspension was incubated with anti-human-CD14(APC) and anti-human CD42b (PE) at RT. Monocytes were determined as CD14brightly positive cells and granulocytes were identified as CD14 dimpositive cells with high side light scattering properties (SSC). Theresults of the first experiment are summarized in Table 1, while Table 2presents average results for two experiments. The data in the Tablesdescribe percentage of leukocytes (granulocytes and monocytes) positivefor each marker in compositions sampled at three time points (FBC, IBCand FP). The CD62L or CD42b positive cells were defined as granulocytesand monocytes with fluorescence greater than that of the same cellsincubated with an appropriate isotype-matching control antibody.

TABLE 1 Expression of cell surface markers indicating leukocyteactivation (representative batch) FBC IBC FP % of CD62L positive cellsMonocytes 68 55 44 Granulocytes 97 47 39 % of CD42b positive cellsMonocytes 26 74 92 Granulocytes 3 15 39

TABLE 2 Average expression of cell surface markers indicating leukocyteactivation FBC IBC FP % of CD62L positive cells Monocytes 64.98 54.7738.285 Granulocytes 97.13 62.44 55.645 % of CD42b positive cellsMonocytes 40.4 68.5 93.0 Granulocytes 3.1 8.8 49.7

As a result of variability between the batches, which depends onindividual characteristics of blood donors, the average of two tests(Table 2) was different from the individual results (Table 1), howeverboth tests and the average data demonstrated similar patterns ofexpression of CD62L and CD42b on monocytes and granulocytes. In the rawmaterial (fresh buffy coat; FBC) percentage of cells expressing CD62Lwas the highest. It decreased during the first incubation (incubatedbuffy coat; IBC) as a result of cell activation and shedding off ofCD62L. Further leukocyte activation during the rest of the productionprocess caused additional loss of CD62L from the cell surface, whichresulted in the lowest percentage of CD62L-positive monocytes andgranulocytes in the final product (FP).

Expression of CD42b had the opposite pattern. The percentage ofCD42b-positive monocytes and granulocytes was lowest in FBC, however itincreased in the IBC because leukocytes became activated and interactedwith platelets membranes containing CD42b. Leukocyte activationcontinued during the rest of the production process resulting in thehighest percentage of CD42b-positive monocytes and granulocytes in theFP.

Thus, as shown in FIGS. 3A and 3B and Tables 1 and 2, comparison ofCD42b and CD62L expression at different stages of the production processdemonstrated increase of the percentage of CD42b and decrease ofCD62L-positive granulocytes and monocytes already at the stage ofIncubated Buffy Coat (IBC) with further respective increase and decreasein the final product (FP), which was in accordance with their activatedstate.

EXAMPLE 2 Analysis of an Activated Leukocyte Composition

Tables 3 and 4 depict the cellular compositions of the final ALCharvested at the end of production as determined by analysis with a CellDyn analyzer. Cells were analyzed (after activated leukocytes werere-suspending suspended the activated leukocytes in serum) was analyzedin triplicate with an automatic Cell Dyn analyzer. In addition cellviability was confirmed by Viable cells were stained using trypan blueexclusion and observed under a microscope. Tables 3 and 4 summarize thecellular composition of 8 batches of the final ALC.

TABLE 3 Composition of Activated Leukocyte Composition PlateletsErythrocytes Leukocytes (10³/μl) (10⁶/μl) (10³/μl) Concentration 46.80.1 6.8 in final ALC Standard 39.2 0.06 3.8 Deviation

TABLE 4 Leukocyte Composition in ALC Leukocytes Granulocytes Neutrophils% Basophils % Eosinophils % Monocytes % Lymphocytes % % in final 65.51.6 4.6 9.1 18.5 ALC Standard  8.2 0.3 3   2.1  4.1 Deviation Range52-78 1-2 1-9 6-12 13-24

EXAMPLE 3 Analysis of an Activated Leukocyte Composition

Leukocytes were sampled at three time points: immediately prior to thebeginning of the production process (fresh buffy coat (FBC); right afterthe first incubation (incubated buffy coat (IBC)); and at the end of theprocess thus producing the final product (FP), meaning afterhypo-osmotic shock, followed by a second, 90 min incubation with serumat 37° C. At each time point, leukocytes were labeled with specificmonoclonal antibodies against corresponding activation markers and thenanalyzed by flow cytometry.

For antibody staining, cells from each time point were washed with FACSstaining solution (PBS, 2% Normal Mouse Serum; 0.02% Sodium Azide),aliquoted at 0.5×10^(6/0) and incubated with appropriate monoclonalantibodies for 30 min. at +4° C. in the dark. After incubation, thecells were treated with erythrocyte lysis buffer, washed, re-suspendedin PBS and analyzed on FACSCalibur flow cytometer (Becton Dickinson).Anti-CD11b and anti-CD62L antibodies were conjugated to phycoerythrin(PE), and CD69 antibody was conjugated to fluorescein isothiocyanate(FITC). In order to better distinguish between leukocyte populations,cells labeled with CD11b were also double-labeled with the antibodyagainst a monocyte marker CD14 conjugated to allophycocyanin (APC), andcells labeled with and CD62L antibodies were also double-labeled withgranulocyte marker CD15-APC. In order to identify T and B lymphocytes,cells stained with CD69 antibody were also double-stained withanti-CD3−APC and anti-CD19-APC antibodies. Cells stained with irrelevantbut isotype-matching antibodies (together with anti-CD14 or anti-CD15,or anti-CD3/CD19 antibodies) under the same conditions were used asnegative controls. Monocytes were determined as CD14 brightly positivecells and granulocytes were identified as CD14 dim positive cells orCD15 bright positive cells with high side light scattering properties(SSC). T Lymphocytes were determined as CD3 positive cells and Blymphocytes were determined as CD19 positive cells. The results of 4-6such individual experiments are summarized in Tables 5, 6 and 7. Thedata are presented as Mean Fluorescence Intensity of stained cellsplus/minus StDev.

The data shown in Table 5 demonstrate that the level of expression ofCD11b recognized by both general (D12) and anti-activated form (CBRM⅕)antibodies significantly increased at the end of the process (FP) on allleukocyte populations compared to Fresh Buffy Coat (FBC). Up-regulationof activated form of CD11b was more pronounced.

TABLE 5 Expression of CD11b on leukocyte populations indicatingleukocyte activation. CD 11b activated form (CBRM CD 11b (D12 antibody)Mean 1/15 antibody) Mean Fluorescence Fluorescence samples GR Mono Tcells B cells GR Mono T cells B cells FBC 635 ± 282 657 ± 104 1.6 ± 1.16.7 ± 3.3 22 ± 13 6 ± 4 0 0.2 ± 0.1 IBC 1099 ± 85  589 ± 12  1.6 ± 1.28.6 ± 5.8 37 ± 6  4 ± 1   0 ± 0.1 0.3 ± 0.2 FP 1100 ± 144  663 ± 48 26.9 ± 21.6 14.3 ± 2.2  225 ± 85  22 ± 8  0.6 ± 0.3 1.5 ± 0.7

TABLE 6 Expression of CD62L on leukocyte populations indicatingleukocyte activation. CD 62L Mean Fluorescence samples GR Mono T cells Bcells FBC 583 ± 428 205 ± 187 256 ± 123 577 ± 165 IBC 34 ± 25 14 ± 8  86± 54 32 ± 19 FP 19 ± 10 16 ± 4  13 ± 6  24 ± 13

TABLE 7 Expression of CD69 on T and B lymphocytes indicating theiractivation. CD69 Mean Fluorescence samples T cells B cells FBC 0.2 + 0.20.2 + 0.1 IBC 0.6 + 0.4 0.6 + 0.4 FP 6.3 + 4.5 8.2 + 3.4

Comparison of CD62L expression at different stages of the productionprocess demonstrated that it drastically and significantly decreased onall leukocyte populations already at the stage of Incubated Buffy Coat(IBC). At the end of the process (FP), CD62L levels were negligible(Table 6).

As shown in Table 7, the upregulation of CD11b and downregulation ofCD62L were consistent with the expression of a specific lymphocyteactivation marker CD69, which increased from practically no expressionto moderate but significant levels.

EXAMPLE 4 Analysis of secretion of IL-8 Cytokine by Activated CellsDuring Incubation With Serum

An activated leukocyte composition containing 10×10⁶ cells was incubatedwith 5 ml of serum in accordance with a preferred embodiment of thepresent invention, and the concentration of IL-8 cytokine was measuredat various time points of incubation using optimized ELISA plates fromeBioscience. Concentrations of IL-8 (pg/ml) in serum without leukocyteswere measured in parallel and subtracted from IL-8 values produced inthe presence of activated leukocytes. The results of five experimentsare summarized as Mean±SD in Table 8.

TABLE 8 Concentrations of IL-8 (pg/ml) released into serum by activatedleukocytes. Time of incubation of ALC with serum 0.5 hour 1 hour 5 hours1115 ± 519 2581 ± 865 12461 ± 9491

The data presented in Table 8 demonstrate sustained release of IL-8 intoserum by ALC for at least 5 hours.

EXAMPLE 5 Identification of Mesenchymal Stem Cells in ALS

ALS was sampled at the end of the production process, doubled-labeledwith specific monoclonal antibodies against pan-leukocyte marker CD45and MSC marker CD105, and then analyzed by flow cytometry.

For antibody staining, cells were washed with FACS staining solution [2%BSA (Sigma), 2% human blocking serum (Chemicon) in DMEM without phenolred (Sigma), pH=7.4] and aliquoted at 100,000 per 100 microL FACS bufferonto a polypropylene U shaped 96 well plate. Each cell aliquot wasincubated with saturating concentrations of antibodies on ice for 30min. At the end of incubation the plate was centrifuged at 200×g for 3min, inverted onto a paper towel to drain the supernatant. The cellswere washed twice in 200 microL FACS buffer and transferred intopolypropylene FACS test tube containing 0.5 ml FACS buffer. The sampleswere kept on ice and in the dark until running on FACSAria (BD).Anti-CD45 antibody was conjugated to Peridinin Chlorophyll ProteinComplex (PerCP) and anti-CD105 antibody was conjugated to fluoresceinisothiocyanate (FITC). The following strategy was used to identify MSCon dot plots. First cells with low side scatter were gated on forwardscatter (FSC-cell size) vs side scatter (SSC-cell granularity) dot plots(FIG. 4A). Then gated cells with low SSC (practically all cells exceptgranulocytes) were plotted against CD105-FITC fluorescence, andCD105^(high) cells were gated (FIG. 4B). Next CD105^(High) cells wereplotted against CD45-PerCP fluorescence, and CD45 negative cells weregated (FIG. 4C). When these cells are plotted in FSC vs SSC coordinates,a quite homogeneous population in terms of size and surface propertiesemerges (FIG. 4D). Statistical analysis of the population of CD45negative/CD105 highly positive cells showed that these cells compose0.2% of all cells in ALS and 2.7% of CD 105 highly positive cells (FIG.4E).

EXAMPLE 6 Identification of Endothelial Progenitor Cells in ALS

ALS was sampled at the end of the production process, doubled-labeledwith specific monoclonal antibodies against CD31, a marker ofendothelial cells and against vascular endothelial growth factorreceptor 2 (VEGFR2, also known as KDR), a marker of early endothelialprogenitors, and then analyzed by flow cytometry.

For antibody staining, cells were washed with FACS staining solution [2%BSA (Sigma), 2% human blocking serum (Chemicon) in DMEM without phenolred (Sigma), pH=7.4] and aliquoted at 100,000 per 100 microL FACS bufferonto a polypropylene U shaped 96 well plate. Each cell aliquot wasincubated with saturating concentrations of antibodies on ice for 30min. At the end of incubation the plate was centrifuged at 200×g for 3min, inverted onto a paper towel to drain the supernatant. The cellswere washed twice in 200 microL FACS buffer and transferred intopolypropylene FACS test tube containing 0.5 ml FACS buffer. The sampleswere kept on ice and in the dark until running on FACSAria (BD).Anti-CD31 antibody was conjugated to phycoerythrin (PE) and anti-KDRantibody was conjugated to allophycocyanin (APC). The results of flowcytometry analysis are shown in FIG. 5. A dot plot graph is depicted onFIG. 5A, showing distribution of stained cells according to theirCD31-PE and KDR-APC fluorescences. The upper right quadrant of the dotplot contains cells double-positive for both markers. Statisticalanalysis showed that this CD31^(positive)/KDR^(positive) cell populationcomposes 0.2% of all cells in ALS. In control experiment an ALS samplewas stained with irrelevant but isotype-matched antibodies. Nopositively-stained cells were found (FIG. 5B).

EXAMPLE 7 Comparison Between Inventive Method and Prior Art Process

To highlight the various unexpected advantages associated with thepresent invention, embodiments thereof were compared to the processdisclosed in U.S. Pat. No. 6,146,890, to Danon (“Danon”).

Inventive Embodiments

For purposes of this example, reference is made to FIGS. 1 and 2illustrated in Applicants' commonly owned International PatentApplication No. PCT/IL2010/000882, filed Mar. 10, 2010 (the disclosureof which is incorporated herein by reference), and the preferredprocedure disclosed therein with respect to use of the 7-bag systemillustrated in those figures. Immediately after separating whole bloodinto its primary components, i.e., red blood cells, plasma, and Buffycoat, the Buffy coat was transferred from Bag C to Bag 4 and incubatedfor 12 hours±2 hours at room temperature. This step was followed bytransferring the distilled water from Bag 1 to Bag 4 (or bag 5), forpurposes of conducting hypo-osmotic shock treatment (resulting in theproduction of a hemolysate). This treatment was conducted forapproximately 45 seconds. Immediately thereafter, the buffered sodiumchloride solution contained in Bag 2 was transferred to Bag 4 (or bag 5)for purposes of restoring isotonicity to the Buffy coat (leukocytes).

Contemporaneous with the incubation of the Buffy coat, above, thebuffered calcium chloride solution in Bag 3 was transferred to Bag Bcontaining the plasma portion, for purposes of allowing for coagulationof the plasma. This was allowed to take place over the course of 12hours±2 hours, plus the additional short time during which theleukocytes were subjected to hypo-osmotic shock and then restoration ofisotonicity.

Immediately following restoration of isotonicity, the entire bagassembly was subjected to centrifugation (typically about 8 to about 10minutes), followed by separation of the cells from the hemolysate. In sodoing, the cells were exposed to hemolysate influence for a minimalperiod of time, i.e., approximately 10 minutes. Followingcentrifugation, the supernatant of the hemolysate in Bag 1 (or bag 5)was discarded, and fresh medium was added to Bag 1 (or bag 5), followedby incubation for about 1-2 hours at 37° C. Following incubation, thecells were subjected to a single wash step.

Comparative (Prior Art) Procedure

Following the teachings in Danon, the Buffy coat was subjected tohypo-osmotic shock immediately after processing the whole blood andseparating it into its 3 main components, i.e., red blood cells, plasma,and the Buffy coat. Thus, in sharp contrast to the present invention,Danon's procedure does not entail incubation of the Buffy coat afterseparation of the main blood components and prior to subjecting theBuffy coat to hypo-osmotic shock. The hypo-osmotic shock was conductedwhile the Buffy coat was contained in Bag PB₃.

As a separate step, and after hypo-osmotic shock the buffered calciumchloride solution was transferred from Bag PB₆ to Bag PB₂ which containsthe plasma, followed by deep-freezing the plasma in PB₂ for 10 minutes,and then placing it in a water bath at 37° C. for an additional 30minute incubation period. During this time, the Buffy coat fractionwhich had been subjected to hypo-osmotic shock was allowed to standwherein the leukocytes remained exposed to the hemolysate.

Following the conclusion of this coagulation process, which in totallasted about 40 minutes, the entire bag system was subject tocentrifugation, and the hemolysate was discarded. Thus, the cells wereexposed to the hemolysate for a period of at least about 55 minutes(i.e., which included the 40-minute standing period that coincided withthe plasma coagulation, and an additional 15 minutes forcentrifugation). In contrast, in the inventive method, the leukocyteswere centrifuged immediately for about 5 min and thus were exposed tohemolysate for much less time, i.e., less than 10 minutes.

After the supernatant from PB₃ was discarded, fresh medium was addedthereto, followed by transferring the entire suspension back to PB_(S),which was then incubated for about 17 hours at 37° C. Followingincubation, the cells were washed 3 times. In contrast, in theembodiments of the present invention, the incubation period wasconducted in coagulated plasma for 1-2 hours, and washed only one time.

Until the aforesaid incubation was conducted, all prior steps werecarried out in transfusion bags, which were the bags the whole blood wascollected in. In contrast, the inventive embodiments entailed use ofinfusion bags which are usually used to administer intravenous solutionssuch as saline.

Danon's procedure was conducted 4 times and compared to the resultsgenerated by the embodiment of the present invention. The results, asset forth below, were averaged.

Results

The total amount of the white blood cells (leukocytes) obtained from astandard blood unit, calculated as a final concentration multiplied byfinal volume of cell suspension, was determined for each batch of wholeblood. The averaged results for the 111 batches of whole blood processedaccording to the method of the present invention, and the 4 batches thatwere processed in accordance with the procedure disclosed in Danon, areset forth in Table 9.

TABLE 9 Yield of WBC per blood unit - (The present invention yielded~90-fold higher amounts of WBCs.) WBC, (×10⁶) Number of batches AverageSD Present invention 449 220 (n = 111) Danon (n = 4) 5 4

The results show that the method of the present invention resulted inabout 90 times greater total number of leukocytes obtained from astandard blood unit (about 450 ml), as compared to Danon's procedure.More generally, the present method results in a yield of at least about100×10⁶, 125×10⁶, 150×10⁶, 175×10⁶, 200×10⁶, 225×10⁶, 250×10⁶, 275×10⁶,300×10⁶, 325×10⁶, 350×10⁶, 375×10⁶, 400×10⁶, 425×10⁶, 450×10⁶, 475×10⁶,500×10⁶, or higher leukocytes per standard blood unit (including allsub-ranges thereof).

The number of viable cells contained within the total cell population,expressed as a percentage, was measured by the Trypan Blue exclusionmethod. The cells were counted in a Newbauer hamemocytometer aftersuspension in Trypan Blue (1:1 ratio) for evaluation of cell count andpercentage of viability. The results are presented in Table 10.

TABLE 10 Viability (% live cells) Procedure Average SD Present invention98% 0.02% (n = 111) Danon (n = 4) 77%   6%

The data in Table 10 show that in addition to the lower yield (as shownin Table 9), Danon's procedure resulted in cell suspension withsignificantly lower viability. That is, almost a quarter (i.e., 23%) ofthe preparation was composed of dead leukocytes (greater than a 10-foldhigher percentage of dead cells). In sharp contrast, almost all of thewhite blood cells processed according to the inventive method weredetermined to be viable. More generally, the inventive ALC may containat least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98% or greater, viable leukocytes based onthis total number of leukocyte cells in the ALC (including allsub-ranges thereof).

Expression of the CD11b activation marker on granulocytes was measuredby flow cytometry, and presented as a percentage of CD11b positive cellsin granulocyte population (CD15 positive cells). Cells sampled fromfinal product were co-stained with anti-CD11b conjugated to fluoresceinisothiocynate (FITC) and anti-CD15 conjugated to phycoerythrin (PE), andanalyzed using FACSCalibur flow cytometer (Becton DickinsonImmunocytometry Systems, San Jose, Calif., USA). CD 11b expression ongranulocytes in cells from 81 final product batches performed inaccordance with the inventive method, and from 3 final product batchesproduced according Danon's procedure, were analyzed.

TABLE 11 CD11b expression on Granulocytes % of Granulocytes expressingCD11b activation marker Procedure Average SD Present invention 84.6 6.2(n = 81) Danon (n = 3) 46.9 9.2

As shown by the data, the inventive method yielded almost two-foldhigher activated granulocytes on a percentage basis as compared toDanon's procedure. More generally, the ALCs of the present invention maycontain at least 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%,61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%,75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, or higher,CD11b(+) granulocytes, relative to the total granulocyte population inthe ALC (including all sub-ranges thereof).

The results of the comparative experimentation demonstrate that thepresently disclosed invention achieves at least 3 unexpected results ascompared to the process disclosed in Danon, namely, a greater yield ofviable leukocytes, a high percentage of viable cells, and a higher levelof granulocyte activation, as shown by higher expression of the CD11bactivation marker. These increases are dramatic and unexpected.

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All publications cited in the specification, including patentpublications and non-patent publications, are indicative of the level ofskill of those skilled in the art to which this invention pertains. Allthese publications are herein incorporated by reference to the sameextent as if each individual publication were specifically andindividually indicated as being incorporated by reference.

Although the invention herein has been described with reference toparticular embodiments, it is to be understood that these embodimentsare merely illustrative of the principles and applications of thepresent invention. It is therefore to be understood that numerousmodifications may be made to the illustrative embodiments and that otherarrangements may be devised without departing from the spirit and scopeof the present invention as defined by the appended claims.

We claim:
 1. A method for making an activated leukocyte compositioncomprising: a) incubating human leukocytes for a period of time so thatthe leukocytes transition from a quiescent to a functionally activestate; b) subjecting the leukocytes to hypo-osmotic shock; and c) addingto the leukocytes of step b) a salt solution in an amount which restoresisotonicity.
 2. The method of claim 1, further comprising mixing theactivated leukocytes of step c) with serum.
 3. The method of claim 1,wherein the incubation of step a) occurs at a temperature of about 12°C. to about 28° C. for a time ranging from about 8 to about 20 hours. 4.The method of claim 3, wherein the incubation of step a) occurs at atemperature of about 18° C. to about 24° C. for a time ranging fromabout 8 to about 12 hours.
 5. The method of claim 1, wherein theincubation of step a) occurs at a temperature of 12° C. to about 28° C.for a time ranging from about 90 minutes, 2 hours, 3 hours, 4 hours, 5hours, 6 hours, 7 hours, 8 hours, or 12 hours to about 20 hours.
 6. Themethod of claim 1, wherein the incubation of step a) occurs at atemperature up to about 37° C. and for a time ranging from five hours toabout 24 hours.
 7. The method of claim 1, wherein the activatedleukocyte composition has a shelf life extending up to 97 hoursfollowing collection of the human leukocytes.
 8. The method of claim 1,wherein the human leukocytes are obtained from a donor having an Onegative blood type.
 9. The method of claim 1, wherein the hypo-osmoticshock comprises contacting the leukocytes with water.
 10. The method ofclaim 2, wherein the serum is prepared from plasma obtained from a donorhaving AB positive blood type.
 11. The method of claim 2, wherein themixture of activated leukocytes and serum is incubated at about 37° C.for about 8-20 hours.
 12. The method of claim 11, wherein the mixture ofactivated leukocytes and serum is incubated for about 60 to about 120minutes.